Glutamate uptake into primary astrocytes was measured using 0.5 µM l-glutamate and 0.3 µCi l-[3H]glutamate per sample (cold:radioactive = 99:1)62. Cells were first washed and pre-incubated at 25°C for 10–20 min in Na+ buffer (5 mM Tris–HCl, pH 7.2, 10 mM HEPES, 140 mM NaCl, 2.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM K2HPO4, and 10 mM d-glucose). Glutamate uptake reaction was initiated by incubating cells for 5 min at 37 °C in Na+ uptake buffer (0.5 µM l-glutamate and 0.3 µCi l-[3H]glutamate per sample in Na+ buffer), followed by two quick washes with ice-cold Na+-free assay buffer (5 mM Tris–HCl, pH 7.2, 10 mM HEPES, 140 mM Choline-Cl, 2.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM K2HPO4, and 10 mM d-glucose). 0.1N NaOH solution was then used to lyse the cells and radioactivity was measured using a scintillation counter. Background radiation was subtracted for each sample separately by incubating the cells for 0 min (immediate removal following addition of hot uptake buffer) on ice followed by quick washes with ice-cold Na+-free assay buffer.
